SMALL NON-CODING RNA REGULATION OF RAS-GTPase FUNCTION in EPIDERMAL HOMEOSTASIS PROJECT SUMMARY/ABSTRACT The Ras-MAPK signal transduction pathway is a critical regulator of the epidermis as dysregulation of Ras- MAPK signaling inhibits epidermal differentiation and is a major driver of tumorigenesis. Our recent discovery that snoRNAs directly interact with and regulate Ras function represents a major paradigm shift in our understanding of small GTPase regulation. Using our novel UV-C cross-linking and immunoprecipitation platform, irCLIP, to characterize transcriptome wide RAS-superfamily GTPase interactions with RNA, we have discovered a rich and complex web of snoRNA-RAS-GTPase interactions suggesting that snoRNAs may regulate all biological processes under RAS-superfamily control, including biochemical signaling nodes, actin/membrane organization, vesicular and intracellular protein trafficking and nuclear/cytoplasmic transport. The long term goals of this K01 application are to deeply characterize the regulatory functions and mechanisms of action of small nucelolar RNAs in modulation of Ras and RAS-superfamily GTPases in control of epidermal homeostasis. In Aim I, we will focus on defining the specificity and breadth of C/D box snoRNA modulation of RAS- superfamily GTPase functions. Our preliminary irCLIP-seq data showed that members of all 5 RAS-subfamilies, RAS, RHO, ARF, RAB and RAN, directly interacted with SNORD50A/B. Thus SNORD50A/B may be a global repressor of RAS-superfamily GTPases as has been described for K-Ras. Using CRISPR/Cas9 gene editing, SNORD50A/B loss-of-function studies will test RAS-GTPase activation levels of 9 RAS-superfamily GTPases spanning all 5 subfamilies. Activation status of biochemical pathways downstream of active-RAS-GTPases will also be monitored with IP-kinase assays and/or phospho-immunoblots when applicable. Our irCLIP-seq data also revealed that Ras isoforms interacted with >20 C/D box snoRNAs, several of which are amplified in cancer. This supports the hypothesis that multiple snoRNAs participate in the regulation of Ras function. In Aim IB, we will use CRISPR-mediated gene editing to excise select Ras-interacting snoRNAs from primary human keratinocytes and assess loss-of-function via analysis of Ras-GTP levels, ERK1/2 and AKT phosphorylation levels, and on epidermal homeostasis in 3D human tissue models. Together, this aim will reveal the extent to which C/D box snoRNAs regulate Ras and RAS-superfamily GTPase functions. Aim II is designed to functionally characterize the RNA-dependent Ras protein interactome. Because of their ability to suppress interaction of Ras with farnesyltransferase, we hypothesized that SNORD50A/B function as adaptors to modulate specific Ras-protein interactions. Using a tandem affinity purification and proximal protein biotinylation (BioID) approach, we compared the interactomes of WT to mutant Ras against WT Ras in a SNORD50A/B +/+ or -/- background. This led to a distilled list of protein interactions that were altered in a SNORD50A/B-specific manner and support the hypothesis that SNORD50A/B regulate vesicular trafficking of Ras from the Golgi to the plasma membrane. CRISPR-mediated loss-of-function studies will be used to assess how these candidate factors contribute to regulation of Ras in control of epidermal homeostasis in 3D tissue models. In a manner orthogonal and complementary to Aim IIA, I will identify global RNA- dependent Ras protein interactions in Aim IIB. SDS-PAGE resolution of irCLIP-adaptor ligated Ras-RNA complexes support the hypothesis that additional proteins are being co-purified with Ras in an RNA-dependent manner. Aim IIB will use our novel irCLIP-mass-spectrometry method to identify these factors. Taken together, this aim will serve to functionally characterize and broaden our understanding of how RNA organizes Ras protein complexes to control epidermal homeostasis.